tbuckley/cabot-search huggingface dataset:
HGG-03. HIGH-GRADE GLIOMA IN YOUNG CHILDREN IS HISTOLOGICALLY, MOLECULARLY, AND CLINICALLY DIVERSE—RESULTS FROM THE SJYC07 TRIAL AND INSTITUTIONAL EXPERIENCE
Authors: Jason Chiang, Aditi Bagchi, Xiaoyu Li, Sandeep K Dhanda, Jie Huang, Soniya N Pinto, Edgar Sioson, Sonia Partap, Paul G Fisher, Daniel C Bowers, Timothy E G Hassall, Congyu Lu, Airen Zaldivar-Peraza, Karen D Wright, Noah D Sabin, Brent A Orr, Arzu Onar-Thomas, Xin Zhou, David W Ellison, Amar Gajjar, Giles W Robinson
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND High-grade gliomas (HGG) in young children pose a challenge due to favorable but unpredictable outcomes. While retrospective studies have broadened our understanding of tumor biology, prospective data regarding outcomes and molecular predictors is lacking. METHODS Young children (0-5 years) histologically diagnosed with HGG and enrolled on the SJYC07 trial or treated at St Jude Children’s Research Hospital from November 2007 to December 2020 were included. DNA methylation, whole genome (WGS), whole exome (WES), and RNA (RNA-seq) sequencing were performed on available samples and these data were integrated with standard histopathological tests to yield a diagnosis. Clinical characteristics and pre-operative imaging were analyzed. RESULTS Fifty-six children (0.0-4.4 years) were identified. Integrated molecular and histopathological analysis split the tumors into four categories: infant-type hemispheric glioma (IHG), HGG, low-grade glioma (LGG), and other-central nervous system (CNS) tumors (i.e., CNS embryonal tumors, CNS sarcomas, neuroepithelial tumors). IHG was the most prevalent (N=22), occurred in the youngest patients (median age 0.4 years; 0-4.4 years), and commonly harbored receptor tyrosine kinase gene fusions (7 ALK, 2 ROS1, 3 NTRK1/2/3, 4 MET). The 5-year event-free (EFS) and overall survival (OS) for IHG was 53.13% (95% CI: 35.52 -79.47) and 90.91% (95%CI: 79.66-100.00) vs. 0.0% and 16.67% (95% CI: 2.78-99.74%) for HGG (p= 0.0043, 0.00013). EFS and OS were not different between IHG and LGG (p=0.95, 0.43). Imaging review of HGG, LGG , IHG and Other CNS tumors showed that IHGs are associated with circumscribed margins (p=0.0047), hemispheric location (p=0.0010), and hemorrhage (p=0.0149). CONCLUSIONS HGG in young children is not a single-entity and is best defined by an integrated histopathological and molecular diagnosis. While patients with IHGs display good survival, as compared to other pediatric HGGs, they still suffer severe treatment-related morbidities. Therefore, prompt consideration for reduced adjuvant and molecularly targeted therapies is warranted.
EPEN-11. CHARACTERIZATION OF THE SPATIAL ARCHITECTURE OF INTRATUMORAL CELL STATES IN PEDIATRIC HIGH-RISK EPENDYMOMA
Authors: Katharina Bruckner, Sibylle Madlener, Carlos A O Biagi Junior, Lisa Gabler, Daniel Senfter, Daeun Jeong, Li Jiang, Bernhard Englinger, Carola Jaunecker, Martin Bilban, Christine Haberler, Christian Dorfer, Karl Rössler, Marcel Kool, Walter Berger, Mariella G Filbin, Daniela Lötsch-Gojo, Johannes Gojo
Journal: Neuro-Oncology (2023)

Abstract By single-cell analyses, we previously uncovered distinct intratumoral transcriptomic ependymoma (EPN) cell states and developmental trajectories correlating with molecular subtype and biological behavior. However, the relation of these cell states to characteristic EPN tissue architecture and tumor microenvironment remains widely unknown. We aimed to characterize the spatial architecture and elucidate regulatory circuits in EPN via spatial transcriptomics (10X Visium) of tumor specimens matching our previously generated single-cell transcriptome data spanning all EPN subtypes (ZFTA, YAP1, PFA, PFB). We investigated whether spatially restricted cell-cell interactions correlate with our previously identified cell states. Analyses revealed distinct spatially resolved cell populations in supratentorial and posterior fossa EPN. All subgroups showed several (5-9) spatially discrete cell clusters. Via non-negative matrix factorization (NMF), cell-specific spatial clusters emerged correlating with previous single-cell programs. ZFTA EPN highly correlates with ZFTA-specific metaprograms whereas all other subgroups did not show ZFTA-specific gene expression. Explicit genes such as DLK1 are exclusively upregulated in ZFTA EPN indicating spatially distinct aberrant Notch pathway signaling. Interestingly, this subgroup revealed spatial zones with upregulation of metabolic genes and endothelial markers anti-correlating with a ZFTA-specific program. Ependymal and neuronal-precursor-like signatures were enriched in our unique YAP1 EPN sample in a spatially restricted pattern, whereas GFAP was ubiquitously expressed. PFA was characterized by a neural stem cell (NSC)-like niche accompanied by metabolic and immune-reactive gene signatures. PFB showed a similar cell niche associated with NSC-like and glial-progenitor-like genes enriched simultaneously, further highlighting the NSC characteristic of posterior fossa tumors. Ependymal signatures were absent in PFA and present in PFB indicating more differentiated cell populations in PFB. Summarizing, we confirm presence of distinct intratumoral cell states within ependymoma tissue and reveal their spatial organization. In depth analysis and functional validation in matched ependymoma models will enable interrogation of underlying tumor promoting signaling events driving intratumoral developmental trajectories.
TRLS-08. EXPERIENCE WITH THE SELECTIVE FIBROBLAST GROWTH FACTOR INHIBITOR ERDAFITINIB IN THREE PEDIATRIC PATIENTS WITH CNS TUMORS
Authors: Natalia Stepien, Lisa Mayr, Cora Hedrich, Maria Aliotti Lippolis, Hannah Schned, Alicia Baumgartner, Adalbert Raimann, Maria-Theresa Schmook, Tatjana Traub-Weidinger, Christian Dorfer, Gabriele Hartmann, Daniel Senfter, Sibylle Madlener, Andreas Peyrl, Amedeo A Azizi, Christine Haberler, Leonhard Müllauer, Johannes Gojo
Journal: Neuro-Oncology (2023)

Abstract Activation of FGF signaling has been described for at least 3% of pediatric solid tumors including pediatric gliomas. Therapeutic actionability of FGF signaling activations, however, remains challenging. Here, we report on three patients with FGFR activation treated with erdafitinib, originally approved for the treatment of FGFR3 or FGFR2 mutated urothelial carcinoma. Our patients were treated for posterior fossa group A ependymoma (patient (Pat) 1&amp;2) and glioma NOS °II-III (Pat3). Dose of erdafitinib at initiation of therapy was 5,2mg/kg/d; 3,8mg/kg/d and 2,8mg/kg/d for Pat1-3 respectively. Rationale for treatment with erdafitinib was increased FGFR3 expression in tumor tissue (TT) of Pat1, increased FGFR1 expression in TT of Pat2, and in Pat3 an activating FGFR1 internal tandem duplication (ITD). Both ependymoma patients received erdafitinib as a fourth line treatment and progressed rapidly under therapy. Treatment was discontinued after 2 weeks (Pat2) and 3 months (Pat1) due to progressive disease. Pat3, with the FGFR1 ITD, responded very well to therapy with a decrease in tumor size and reduced contrast-enhancement after 3&amp;6 months. Therapy was discontinued after six months due to dystrophic nail changes. During treatment, our patients reported on diarrhea, abdominal pain, pain in the lower extremities, dysgeusia, hypersensitivity of the oral mucosa, dry skin and dystrophic nail changes. Additionally, we observed a distinct increase in longitudinal growth (Pat1&amp;3), increased serum phosphate levels, LFPs, alpha-amylase, cholinesterase, alkaline phosphatase and LDH. Summing up, treatment with erdafitinib has high potential for a selected cohort of pediatric CNS tumor patients, as demonstrated by the treatment response of Pat3 with the FGFR1 IDT. However, further defining the exact molecular alterations that predict the individual patient’s response as well as side effects in the pediatric population are imperative to elucidate the further role of erdafitinib in treatment of pediatric CNS tumors.
DIPG-37. TRANSCRIPTIONAL, EPIGENETIC, AND IMMUNOLOGICAL FEATURES OF PAEDIATRIC-TYPE H3 K27-ALTERED DIFFUSE MIDLINE GLIOMAS ORIGINATING FROM DIFFERENT BRAIN LOCATIONS
Authors: Yura Grabovska, Alan Mackay, Rita Pereira, Drenusha Sejdiu, Anna Burford, Sara Temelso, Matt Clarke, Chris Jones
Journal: Neuro-Oncology (2023)

Abstract Diffuse midline gliomas (DMG) are highly aggressive brain tumours predominantly affecting young children. The main oncogenic driver is the substitution of lysine 27 to methionine (K27M) in histone 3 (H3) or overexpression of EZHIP resulting in global loss of H3K27me3. Recent single-cell (epi-)genomic studies have identified developmentally-restricted origins and cell states associated with tumour location and patient age, and we aimed to further explore the underlying biology of distinct subgroups of DMG using orthogonal data modalities. A retrospective reanalysis identified 242 patient genome-wide DNA methylation array profiles with a methylation classification of DMG_K27. H3 status was known for 168 cases (H3.3 K27M n=130, H3.1 K27M n=20, H3 WT n=18), with EZHIP overexpression or loss of H3K27me3 was confirmed in 13/18 H3 WT cases. Unsupervised clustering identified two distinct methylation subtypes, specific to brainstem (62/64) or thalamic (25/29) location, and highly enriched for gain of chromosome 1q or loss of 5q, respectively. Differential expression analysis from bulk RNA-Seq data recapitulated the recently-published overexpression of ZIC1/4 early development genes in thalamic tumours and an enrichment of HOX-cluster genes in those of brainstem origin. Within DMGs of the thalamus, we further observed a significant overexpression and hypomethylation of NR2E1, a regulator of bivalent promoters via LSD1-H3K4, and essential for stem cell renewal; and CD96, an immune checkpoint blockade target in NK/T-cells. We confirmed differential immune response expression between tumour location by GSEA as well as deconvolution by methylation CIBERSORT from bulk samples, which we were able to characterise further by by single-cell RNA-sequencing. We report unique copy-number and differentially methylated loci as well as differential expression that recapitulates previously reported single-cell metaprogrammes. We also highlight involvement of neural stem cell development programme in thalamus-type tumours as well as evidence of these tumours as being potential targets for immune therapeutic targeting.
DIPG-34. IMMUNE SYNAPSE OPTIMIZED GRP78-SPECIFIC CAR T CELLS ELICIT AN IMPROVED ANTITUMOR RESPONSE AGAINST DIPGS
Authors: Jorge Ibanez, Haley Houke, Michaela Meehl, Nikhil Hebbar, Unmesha Thanekar, Paulina Velasquez, Giedre Krenciute
Journal: Neuro-Oncology (2023)

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors and a leading cause of cancer-related death in children. There is currently no cure for DIPG, highlighting an urgent need for novel therapeutics. Chimeric antigen receptor (CAR)-engineered T-cell therapy offers great promise for DIPG treatment; however, several limitations need to be addressed. In this study, we targeted a novel tumor associated antigen GRP78 after showing it is reliably expressed on the cell surface of DIPGs. To test the anti-DIPG activity of GRP78-CAR T cells, we evaluated cytotoxicity, persistence, and cytokine secretion in vitro. We found that GRP78-CAR T cells kill DIPGs but fail to proliferate and secrete cytokines compared to positive control, U87. In vivo studies further confirmed that GRP78-CAR T cells eradicated U87 tumors but not DIPGs. Based on these findings, we concluded that DIPGs cannot trigger efficient CAR T cell signaling. Because CAR T cell effector function is influenced by antigen density, we quantified the relative GRP78 expression on DIPGs and other brain tumors, including U87. We found that GRP78 is comparatively lowly expressed on DIPGs. Live-cell imaging of calcium flux in CAR T cells further revealed that T cell activation is lower upon interaction with DIPGs than U87s, suggesting that CAR-T cells form incomplete or dysfunctional immune synapses (IS) against DIPGs. To improve IS function, we knocked-out RASA2, a TCR downstream signaling inhibitor. Our results show that T cells lacking RASA2 have an improved IS reflected by bigger synaptic areas, increased lytic granules, and pZAP70 accumulation at the synapse. RASA2 KO further increased calcium influx and proliferation capacity of GRP78-CAR T cells against DIPGs. Thus, this study confirms that antigen density controls CAR T cell antitumor activity, and improving the IS formation of CAR T cells elicits enhanced antitumor response against low antigen-expressing tumor cells.
DIPG-49. A SYSTEMS BIOLOGY APPROACH TO DEFINING AND TARGETING CELL STATE-SPECIFIC MASTER REGULATOR DEPENDENCIES IN DIFFUSE MIDLINE GLIOMA
Authors: Ester Calvo Fernández, Junqiang Wang, Xu Zhang, Hong-Jian Wei, Hanna Minns, Aaron Griffin, Aleksandar Obradovic, Lukas Vlahos, Timothy Martins, Pamela Becker, John Crawford, Robyn Gartrell, Luca Szalontay, Stergios Zacharoulis, Zhiguo Zhang, Robert Wechsler-Reya, Cheng-Chia Wu, Andrea Califano, Jovana Pavisic
Journal: Neuro-Oncology (2023)

Abstract Diffuse Midline Glioma (DMG) are fatal pediatric brain tumors. We leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct cell states. We produced a DMG regulatory network from 122 publicly available RNAseq profiles with ARACNe, and inferred sample-specific MR protein activity with VIPER. A CRISPR/Cas9 KO screen across 3 DMG patient cell lines identified a set of 73/77 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p=0.000034). FOXM1 emerged as an essential MR, significantly activated across virtually all patients. We then generated RNAseq profiles following perturbation with ~300 oncology drugs in 2 DMG cell lines most representative of patient MR signatures, and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Dept.of Health approved OncoTreat algorithm. OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and proteosome inhibitors in subsets of patients. 80%of OncoTreat-predicted drugs (p&amp;lt;10-5) from 3 DMG patient tumor biopsies showed in vitro sensitivity in cultured tumor cells from the respective patients, with overall 68% accuracy among 223 drugs evaluated by both OncoTreat and in vitro (Fisher’s Exact Test p=0.0449). Further analysis of DMG intra-tumor heterogeneity via protein activity inference from published scRNAseq profiles identified 6 tumor clusters with unique MR signatures representing distinct cellular states. Targetable MRs and OncoTreat-predicted drugs were distinct between these states. Bulk RNAseq analysis recapitulated predictions seen in the more prevalent Oligodendrocyte progenitor cell-like states, but failed to capture MR and drug predictions for the Astrocyte-like states. Ongoing validations of cell state-specific drug predictions in vivo in subcutaneous patient-derived xenograft and orthotopic syngeneic DMG models have already shown tumor volume and subpopulation differences (e.g. Trametinib-treated). This provides a platform to nominate much-needed novel drugs to treat DMG.
DIPG-08. THE LANDSCAPE OF TUMOR CELL STATES AND SPATIAL ORGANIZATION IN H3-K27M MUTANT DIFFUSE MIDLINE GLIOMA ACROSS AGE AND LOCATION
Authors: Ilon Liu, Li Jiang, Erik Samuelsson, Sergio Marco Salas, Alexander Beck, Olivia Hack, Daeun Jeong, McKenzie Shaw, Bernhard Englinger, Jenna LaBelle, Hafsa Mire, Sibylle Madlener, Lisa Mayr, Michael Quezada, Maria Trissal, Eshini Panditharatna, Kati Ernst, Jayne Vogelzang, Taylor Gatesman, Matthew Halbert, Hana Palova, Petra Vesela, Jaroslav Sterba, Ondrej Slaby, Rene Geyeregger, Aaron Diaz, Izac Findlay, Matt Dun, Adam Resnick, Mario Suva, David Jones, Sameer Agnihotri, Jessica Ostlin, Carl Koschmann, Christine Haberler, Thomas Czech, Irene Slavc, Jennifer Cotter, Keith Ligon, Sanda Alexandrescu, W K Alfred Yung, Isabel Arrillaga-Romany, Johannes Gojo, Michelle Monje, Mats Nilsson, Mariella Filbin
Journal: Neuro-Oncology (2023)

Abstract Histone 3 lysine27-to-methionine mutations (H3-K27M) frequently occur in childhood diffuse midline gliomas (DMGs) of the pons, thalamus, and spinal cord, presumed to be driven by the specific spatiotemporal context of these midline locations during postnatal development. While most common in the pons and at mid-childhood ages, the same oncohistone mutation is recurrently detected in adult DMGs and throughout different midline regions. The potential heterogeneity of tumors at different ages and in different anatomical locations of the midline are vastly understudied. Through dissecting the transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs - spanning the age range from 2-68 years and locations from spinal cord to thalamus - at single cell resolution, we delineate how age- and location-dependent contexts shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We identify that oligodendrocyte precursor (OPC)-like cells constitute the stem-like compartment in H3-K27M DMGs across all clinico-anatomical groups, however, depending on location, display varying levels of maturity resembling less differentiated pre-OPCs or more mature OPCs further differentiated along the oligodendroglial lineage. We further demonstrate increased mesenchymal cell states in adult tumors, which we link to age-related differences in glioma-associated immune cell compartments, in particular an increase of macrophages in adult compared to pediatric tumors. Furthermore, we resolve the spatial organization of H3-K27M DMG cell types and states in intact patient tissues, identifying a local niche of the oligodendroglial lineage. Our study provides a powerful resource for rational modeling and therapeutic frameworks taking into account determinants of age and location in this lethal glioma group.
DIPG-42. MAPPING THE DNA METHYLATION LANDSCAPE OF DIPG REVEALS NOTABLE METHYLOME VARIABILITY RESPONSIVE TO PHARMACOLOGIC MODULATION
Authors: Ashley Tetens, Allison Martin, Antje Arnold, Orlandi Novak, Adrian Idrizi, Rakel Tryggvadottir, Jordyn Craig-Schwartz, Michael Barbato, Charles Eberhart, Adam Resnick, Michael Koldobskiy
Journal: Neuro-Oncology (2023)

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.
EPEN-12. COMPARING CELLULAR HIERARCHIES AND SPATIAL ARCHITECTURE ACROSS MOLECULAR SUBGROUPS OF SUPRATENTORIAL EPENDYMOMAS
Authors: Daeun Jeong, Kendra K Maass, David R Ghasemi, Bernhard Englinger, Li Jiang, Andrezza Nascimento, Olivia A Hack, McKenzie L Shaw, Stefan M Pfister, Marcel Kool, Johannes Gojo, Kristian W Pajtler *, Mariella G Filbin *
Journal: Neuro-Oncology (2023)

Abstract Ependymomas (EPN) are among the most common and fatal pediatric brain tumors with currently no targeted therapies available. Molecular studies including genome-wide DNA methylation profiling have shown that supratentorial ependymoma is subdivided into subgroups characterized by distinct fusion proteins: ZFTA-RELA fusion-positive (ZFTA-RELA), YAP1 fusion-positive (ST-YAP1), and four alternative ZFTA fusion-positive (ZFTA-Cluster 1, ZFTA-Cluster 2, ZFTA-Cluster 3, and ZFTA-Cluster 4). ZFTA-RELA tumors have been identified as the most common and aggressive molecular group with high intra-tumoral heterogeneity. However, limited sample size and interpatient variability in previous studies highlight a critical need for comprehensive profiling of supratentorial ependymomas across a larger patient cohort. In particular, how ZFTA-RELA compares to other subgroups of supratentorial ependymoma is unknown. Here, we collected and profiled 44 supratentorial patient tumors encompassing ZFTA-RELA (n=21), ZFTA-Cluster 1 (n=6), ZFTA-Cluster 2 (n=4), ZFTA-Cluster 3 (n=7), ZFTA-Cluster 4 (n=2), and ST-YAP1 (n=4). We profiled all tumors by single cell/single nucleus RNA-sequencing, and further characterized ZFTA-RELA by single cell spatial transcriptomics. Within each patient’s tumor we find multiple normal and aberrant differentiation trajectories from neural progenitor-like cells towards glial-like, mesenchymal-like and ependymal-like cells. Using sequencing-based lineage tracing, we experimentally validate these computationally inferred cellular hierarchies in vitro. Notably, compared to ZFTA-RELA ependymomas we observed predominantly higher proportion of early neural progenitor-like cells in ZFTA-Cluster 2 and ZFTA-Cluster 3, while in ST-YAP1 greater representation of mature ependymal-like cells. These findings highlight differences in developmental and aberrant differentiation trajectories driven by combination of oncogenic fusions and cell of origin in supratentorial ependymoma subgroups. In addition, neural progenitor-like cells most highly express ZFTA-fusions and are spatially distinct from other cell states, highlighting oncogenic fusions driving tumorigenesis organized in spatial niches. Together, this study reveals previously unknown intra- and intertumoral heterogeneity across subgroups of supratentorial ependymoma, as well as distinct cellular and spatial architecture.